摘要
将完整的pAPN序列通过同源重组克隆入慢病毒表达载体pLVX-mCherry,并将该重组质粒pLVX-mCherry-pAPN与慢病毒包装质粒pLP1、pLP2、pLP-VSVG共转染293T细胞,在293T细胞中包装成含目的基因的重组慢病毒。将收获的重组病毒感染Vero细胞,经过嘌呤霉素筛选和细胞有限稀释法,筛选出表达pAPN的细胞。通过RT-PCR和间接免疫荧光试验(IFA)证明了所获细胞株能够稳定表达pAPN蛋白,命名为Vero-APN-B6。IFA试验和TCID50试验结果表明:与Vero细胞相比,Vero-APN-B6更能促进猪流行性腹泻病毒(PEDV)的增殖。利用该细胞株成功地从猪小肠组织中分离到了PEDV SC-MS株;经过鉴定该毒株为变异毒株。
The porcine aminopeptidase N sequence was cloned into the lentiviral vector p LVX-m Cherry via homologous recombination.The recombinant plasmid p LVX-m Cherry-pAPN was transfected into 293 T with lentiviral packaging plasmids PLP1, plp2, PLP/VSVG,respectively.The recombinant virus was harvested and Vero cells were infected.The cells expressing p APN were selected by culturing in nutrient solutions containing puromycin and limitied dilution. The expression of APN in the cell line was identified by RT-PCR and IFA. A monoclonal cell line was successfully obtained using single cell cloning technology and,was named Vero-APN-B6.The susceptibility to porcine epidemic diarrhea virus（PEDV） of this cell line,compared with the normal Vero cells,was detected by the methods of IFA and determination of TCID50.The results showed that the cell line was more susceptible to PEDV than the normal Vero cells.PEDV SC-MS stain was isolated from small intestinal tissues using this cell line.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2017年第4期419-426,共8页
Chinese Veterinary Science
基金
南京农业大学科研项目(KJQN201619)
关键词
pAPN
细胞系
慢病毒载体
PEDV
SC-MS株
porcine aminopeptidase N
cell line
lentiviral vector
porcine epidemic diarrhea virus
SC-MS stain
作者简介
陆倩倩（1991-），女，江苏南通人，硕士生，研究方向为动物传染病预防与免疫。
通信作者：王先炜，副教授，硕士生导师，主要从事动物传染病的教学与研究工作。E-mail：xwwang@njau．edu．cn．
